The glycoprotein hormone erythropoietin regulates the growth and differentiation of red blood cell (erythrocyte) progenitors. The hormone is produced in the fetal liver and adult kidney. Erythropoietin induces proliferation and differentiation of red blood cell progenitors through interaction with receptors on the surface of erythroid precursor cells.
Several approaches have been employed to identify those features of the protein that are relevant to its structure and function. Examination of the homologies among the amino acid sequences of erythropoietin proteins of various species has demonstrated several highly conserved regions (McDonald, J. D., et al., Mol. Cell. Biol. 6: 842-848 (1986)).
Oligonucleotide-directed mutagenesis has been used to prepare structural mutants of erythropoietin, lacking specific sites for glycosylation. Studies indicate that N-linked carbohydrates are important for proper biosynthesis and/or secretion of erythropoietin. These studies also show that glycosylation is important for in vivo, but not in vitro, biological activity. (Dube, S., et al., J. Biol. Chem. 263:17516-17521 (1988); Yamaguchi, K., et al., J. Biol. Chem. 266:20434-20439 (1991); Higuchi, M., et al., J. Biol. Chem. 267:7703-7709 (1992)).
Studies with monoclonal anti-peptide antibodies have shown that the amino terminus and the carboxy-terminal region (amino acids 152-166) of erythropoietin may be involved with biological activity. It has also been demonstrated that antibodies to amino acids 99-119 and 111-129 block the hormone's biological activity, apparently by binding to two distinct non-overlapping domains (99-110 and 120-129). (Sytkowski, A. J. and Donahue, K. A., J. Biol. Chem. 262:1161-1165 (1987)). Thus, it was hypothesized that amino acids 99-129 were important in the formation of a functional region involved in receptor recognition, either through forming a necessary component of the protein's tertiary structure or through direct participation in receptor binding, or both.
Preliminary experiments suggested that alterations in localized secondary structure within the 99-129 region resulted in inactivation of erythropoietin. Therefore, a possible structural role for amino acids 99-129 has been postulated. Recently, a series of experiments indicated that amino acids 99-110 (Domain 1) play a critical role in establishing the biologically active conformation of human erythropoietin. (Chern, Y., et al., Eur. J. Biochem. 202:225-229 (1991)).
These Domain 1 mutants, in which a group of three amino acids was deleted and replaced by two different amino acids, were found to be biologically inactive. Furthermore, these mutations in Domain 1 inhibited the secretion of the mutant erythropoietin into cell culture medium. (Chern, Y., et al., Eur. J. Biochem. 202:225-229 (1991)). Inhibition of secretion in mammalian cells is consistent with a profound structural change of the polypeptide hormone. Profound structural changes could significantly affect the ability of the hormone to interact with its cognate receptor. Thus, these mutant erythropoietin polypeptides are not suitable for elucidating the structure/function relationship that exists between erythropoietin and its cellular receptor. Nor are these mutants suitable erythropoietin antagonists for use, for example, in therapeutic treatment of polycythemias, or over production of erythropoietin. Thus, it would be beneficial to precisely determine which amino acids are critical to the erythropoietin polypeptide to maintain a stable, biologically active conformation which retains its secretable properties and its ability to bind to the erythropoietin receptor.
Moreover, the precise determination of critical amino acid residues would be useful to alter the biological activity of erythropoietin, either decreasing or increasing one or more biological properties of the protein.